A simple aspirin-inducible system has been developed and characterized in Escherichia coli by employing the Psal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that the effector-free form, SalRr, can compete with the effector-bound form, SalRa, binding the Psal promoter and repressing gene transcription. The induction of the Psal promoter was compared in two different gene circuit designs: a simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of 0.05 to 10 µM aspirin. Overexpression of SalR in the SRS circuit reduced both baseline leakiness and the strength of the Psal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on the SalRr/SalRa competitive binding model not only fit the observed experimental results in SRS and PAR circuits but also predicted the performance of a new gene circuit design for which weak expression of SalR in the SRS circuit should significantly improve induction strength. The experimental result is in good agreement with this prediction, validating the SalRr/SalRa competitive binding model. The aspirin-inducible systems were also functional in probiotic strain E. coli Nissle 1917 and SimCells produced from E. coli MC1000 ΔminD. These well-characterized and modularized aspirin-inducible gene circuits would be useful biobricks for synthetic biology. IMPORTANCE An aspirin-inducible SalR/Psal regulation system, originally from Acinetobacter baylyi ADP1, has been designed for E. coli strains. SalR is a typical LysR-type transcriptional regulator (LTTR) family protein and activates the Psal promoter in the presence of aspirin or salicylate in the range of 0.05 to 10 µM. The experimental results and mathematical simulations support the competitive binding model of the SalR/Psal regulation system in which SalRr competes with SalRa to bind the Psal promoter and affect gene transcription. The competitive binding model successfully predicted that weak SalR expression would significantly improve the inducible strength of the SalR/Psal regulation system, which is confirmed by the experimental results. This provides an important mechanism model to fine-tune transcriptional regulation of the LTTR family, which is the largest family of transcriptional regulators in the prokaryotic kingdom. In addition, the SalR/Psal regulation system was also functional in probiotic strain E. coli Nissle 1917 and minicell-derived SimCells, which would be a useful biobrick for environmental and medical applications.
Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies indicate that a single clock gene may determine the temperature compensation in several model organisms. In order to understand the influence of each individual clock gene on the temperature compensation, twenty-three well-known mammalian clock genes plus Timeless and Myc genes were knocked out individually, using a powerful gene-editing tool, CRISPR/Cas9. First, Bmal1, Cry1, and Cry2 were knocked out as examples to verify that deleting genes by CRISPR is effective and precise. Cell lines targeting twenty-two genes were successfully edited in mouse fibroblast NIH3T3 cells, and off-target analysis indicated these genes were correctly knocked out. Through measuring the luciferase reporters, the circadian periods of each cell line were recorded under two different temperatures, 32.5 °C and 37 °C. The temperature compensation coefficient Q10 was subsequently calculated for each cell line. Estimations of the Q10 of these cell lines showed that none of the individual cell lines can adversely affect the temperature compensation. Cells with a longer period at lower temperature tend to have a shorter period at higher temperature, while cells with a shorter period at lower temperature tend to be longer at higher temperature. Thus, the temperature compensation is a fundamental property to keep cellular homeostasis. We further conclude that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. We also estimated the proliferation rates of these cell lines. After systematically comparing the proliferation rates and circadian periods, we found that the cell growth rate is not dependent on the circadian period.
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